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Peer-reviewedCC BYBioinformatics

Barbell reveals and resolves demultiplexing and trimming issues in Nanopore data

  • Rick Beeloo1
  • Ragnar Groot Koerkamp2
  • Xiu Jia3
  • Marian J. Broekhuizen‐Stins4
  • Lieke van Ijken4
  • Els M. Broens4
  • Aldert Zomer4
  • Bas E. Dutilh1,3
4 affiliations
  1. 1Department of Biology, Science4Life, Utrecht University , Utrecht, 3584 CH,
  2. 2Department of Computer Science, Karlsruhe Institute of Technology , Karlsruhe, 76131,
  3. 3Institute of Biodiversity, Ecology, and Evolution, Faculty of Biological Sciences, Cluster of Excellence Balance of the Microverse, Friedrich Schiller University Jena , Jena, 07743,
  4. 4Division of Infectious Diseases and Immunology, Utrecht University , Utrecht, 3508 TD,
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Abstract

MOTIVATION: Oxford Nanopore sequencing enables long-read analysis for diverse applications, but artefacts introduced by Nanopore barcoding are poorly characterized and can compromise demultiplexing accuracy and downstream analyses. RESULTS: Using a rapid barcoding experiment on 66 diagnostic samples, we found that only 83% of reads followed the expected single-barcode configuration, while 17% showed complex barcode attachments. We observed similar patterns in public datasets, and also in native barcoding datasets where only 30%-70% of the reads had barcodes on both ends. Widely used demultiplexers, including Dorado, fail to resolve these cases, leaving ∼10% of our rapid barcoding reads partially trimmed and contaminated with adapter fragments. We developed Barbell, a pattern-aware demultiplexer that is designed to detect complex barcode configurations. Barbell reduced contaminated reads from >400 000 (Dorado/Flexiplex) to 166 (99.96% reduction), minimized barcode bleeding, and supports custom experimental designs such as dual-end barcodes and shorter barcodes (e.g. Illumina barcodes). We further show that such contamination is widespread in public databases, with Nanopore sequences detected in hundreds of NCBI entries, some of which are responsible for artificial taxonomic connections. AVAILABILITY AND IMPLEMENTATION: Barbell is open source and available at https://github.com/rickbeeloo/barbell.

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Cite

BibTeXRIS
BibTeX
@article{beeloo2026barbell,
  title = {{Barbell reveals and resolves demultiplexing and trimming issues in Nanopore data}},
  author = {Rick Beeloo and Ragnar Groot Koerkamp and Xiu Jia and Marian J. Broekhuizen‐Stins and Lieke van Ijken and Els M. Broens and Aldert Zomer and Bas E. Dutilh},
  journal = {Bioinformatics},
  year = {2026},
  month = may,
  doi = {10.1093/bioinformatics/btag349},
  url = {https://doi.org/10.1093/bioinformatics/btag349},
  note = {CC BY},
}
RIS
TY  - JOUR
TI  - Barbell reveals and resolves demultiplexing and trimming issues in Nanopore data
AU  - Rick Beeloo
AU  - Ragnar Groot Koerkamp
AU  - Xiu Jia
AU  - Marian J. Broekhuizen‐Stins
AU  - Lieke van Ijken
AU  - Els M. Broens
AU  - Aldert Zomer
AU  - Bas E. Dutilh
PY  - 2026
DA  - 2026/05/28
JO  - Bioinformatics
DO  - 10.1093/bioinformatics/btag349
UR  - https://doi.org/10.1093/bioinformatics/btag349
AB  - MOTIVATION: Oxford Nanopore sequencing enables long-read analysis for diverse applications, but artefacts introduced by Nanopore barcoding are poorly characterized and can compromise demultiplexing accuracy and downstream analyses. RESULTS: Using a rapid barcoding experiment on 66 diagnostic samples, we found that only 83% of reads followed the expected single-barcode configuration, while 17% showed complex barcode attachments. We observed similar patterns in public datasets, and also in native barcoding datasets where only 30%-70% of the reads had barcodes on both ends. Widely used demultiplexers, including Dorado, fail to resolve these cases, leaving ∼10% of our rapid barcoding reads partially trimmed and contaminated with adapter fragments. We developed Barbell, a pattern-aware demultiplexer that is designed to detect complex barcode configurations. Barbell reduced contaminated reads from >400 000 (Dorado/Flexiplex) to 166 (99.96% reduction), minimized barcode bleeding, and supports custom experimental designs such as dual-end barcodes and shorter barcodes (e.g. Illumina barcodes). We further show that such contamination is widespread in public databases, with Nanopore sequences detected in hundreds of NCBI entries, some of which are responsible for artificial taxonomic connections. AVAILABILITY AND IMPLEMENTATION: Barbell is open source and available at https://github.com/rickbeeloo/barbell.
SN  - 1367-4803
SN  - 1367-4811
ER  -